Synergistic effect of Salvadora persica and chitosan nanoparticles against oropharyngeal microorganisms

Herbal medicine combined with nanoparticles has caught much interest in clinical dental practice, yet the incorporation of chitosan with Salvadora persica (S. persica) extract as an oral care product has not been explored. The aim of this study was to evaluate the combined effectiveness of Salvadora persica(S. persica) and Chitosan nanoparticles (ChNPs) against oropharyngeal microorganisms. Agar well diffusion, minimum inhibitory concentration, and minimal lethal concentration assays were used to assess the antimicrobial activity of different concentrations of ethanolic extracts of S. persica and ChNPs against selected fungal strains, Gram-positive, and Gram-negative bacteria. A mixture of 10% S. persica and 0.5% ChNPs was prepared (SChNPs) and its synergistic effect against the tested microbes was evaluated. Furthermore, the strain that was considered most sensitive was subjected to a 24-h treatment with SChNPs mixture; and examined using SEM, FT-IR and GC–MS analysis. S. persica extract and ChNPs exhibited concentration-dependent antimicrobial activities against all tested strains. S. persica extract and ChNPs at 10% were most effective against S. pneumoni, K. pneumoni, and C. albicans. SEM images confirmed the synergistic effect of the SChNPs mixture, revealing S. pneumonia cells with increased irregularity and higher cell lysis compared to the individual solutions. GC–MS and FT-IR analysis of SChNPs showed many active antimicrobial phytocompounds and some additional peaks, respectively. The synergy of the mixture of SChNPs in the form of mouth-rinsing solutions can be a promising approach for the control of oropharyngeal microbes that are implicated in viral secondary bacterial infections.

The mouth is the main gateway to the body and contains a highly diverse microbiota, most of which are commensal.Under normal conditions, the microbiota is able to migrate from the oral cavity to the lungs; hence, the lungs are enriched with oral taxa 1 .El-Solh et al. 2 demonstrated that respiratory pathogens from the lung of institutionalized elder patients requiring mechanical ventilation are often genetically indistinguishable from strains isolated from the oral cavity.Co-infections caused by bacteria or fungi in several influenza virus pandemics have increased nowadays, and bacterial complications have increased the morbidity and mortality of viral infections, including Covid-19 3 .
S. pneumoniae was isolated in almost 50% of the cases of hospital-acquired pneumonia 4 .It is the most common bacteria found in viral secondary bacterial infections and is mostly associated with high mortality and morbidity during influenza epidemics and pandemics 5 .A recent investigation demonstrated that poor oral hygiene was linked to an increase in the number of obligate anaerobes located in the pneumonia-affected lungs of the studied patient population 6 .Professional oral hygiene measures decrease the number of oral bacteria 7 , reduce the number of days of fever, inhibit the development of pneumonia 8 , reduce the frequency of nosocomial pneumonia by nearly 40% 9 , and reduce the mortality rate caused by pneumonia 10 .Several other oral care practices have been attempted, including tooth brushing and mouthwashes.Gargling is considered to have promising effects through the removal of oral/pharyngeal proteases that help viral replication 11 .
Salvadora persica (S. persica), locally called miswak, is a traditional toothbrush that belongs to the Salvadoraceae family and has demonstrated antimicrobial properties against bacteria, viruses, and fungi [12][13][14] .The antiinflammatory and antimicrobial effects of S. persica extracts on cariogenic and periodontal pathogens led to their incorporation into oral hygiene products such as mouthwashes and toothpastes 15 .Almaghrabi 16 demonstrated strong activity of extracts obtained from fruits, twigs, and roots of S. persica against S. pneumoniae wild-type strains.Recent findings showed that S. persica extract decreased the amount of oral pathogen colonization in mechanically ventilated patients 17 .
In recent years, nanotechnology has emerged as an interdisciplinary field undergoing rapid development as a powerful tool for various biomedical applications, especially as antimicrobial agents 18 .Many oral care products, especially mouthwash and toothpastes, contain nanoparticles with anti-microbial, anti-inflammatory, and remineralizing properties 19 have been investigated in dental applications.Among the various existing nanomaterials, chitosan nanoparticles (ChNPs) showed excellent antibacterial, antiviral, and antifungal properties 20 .
Combining S. persica extract with ChNPs is a novel approach with the intent of producing a synergistic antimicrobial effect against a wide range of microorganisms.Since there is evidence suggesting a close relationship between ventilator-associated pneumonia and the microbial profile present in the oral cavity 21 , this combination might be used as a preventive measure to control potentially harmful oropharyngeal microorganisms, as well as a therapeutic agent for established infections.
The incorporation of ChNPs with S. persica extract as an oral care product has not been extensively explored.Therefore, the aim of this study was to evaluate the antimicrobial efficacy of the ethanolic S. persica extract and ChNPs mixture against oropharyngeal microorganisms.

Salvadora persica extract preparation
The plant collection and use were in accordance with all the relevant guidelines.S. persica root sticks were obtained from the Jazan region of Saudi Arabia.The sticks were cut into small pieces and milled by an electric grinder.Fifty grams of S. persica powder were extracted by soaking in 500 mL of 70% ethanol on a shaker at room temperature for 24 h.The extract solutions were then filtered using Whatman No. 4 filter paper and transferred to a porcelain dish to evaporate at 40 °C.The solutions were completely evaporated, and the powdered extract was stored in a refrigerator (4 °C) until required.

Chitosan nanoparticles (ChNPs)
Chitosan nanoparticles were purchased from Nanoshel-UK Ltd. and prepared using the ionic gelation method.Different concentrations of chitosan (0.1 to 0.5 g) were added to 100 mL of 1% l-ascorbic acid and mixed well using a magnetic stirrer.The microbial strain was cultured on freshly prepared tryptic soy broth (TSB) (OXOID, Hampshire, England) for 18 h at 36 ± 1 °C to an exponential phase.Streptococcus species were additionally cultured on Brain Heart Infusion Broth (BHIB) (Difco, Laboratories, Detroit, MI) with 5% CO 2 and incubated at 35 ± 1 °C.The optical density (OD 620 ) of the microbial mass was detected by determining the turbidity of the bacterial growth using a spectrophotometer.The inoculum density was adjusted using normal saline to produce a final count of 1.5 × 10 6 CFU/mL.The stock solution of S. persica extract was dissolved in dimethyl sulfoxide (DMSO) to achieve a final concentration of 512 mg/mL.

Antimicrobial testing
The antimicrobial activity of different concentrations (25, 50, and 100 mg/mL) of S. persica extract and ChNPs was evaluated using standard agar well diffusion with Streptomycin (30 μg) and Amphotericin B (10 μg) discs (Oxoid, UK) serving as positive controls against bacterial and fungal strains, respectively, while ethanol and propylene glycol solution served as a negative controls for S. persica extract and ChNPs, respectively.All experiments were conducted in triplets.
Minimum inhibitory concentration (MIC) was performed by a standard twofold broth dilution method.Chlorohexidine (CHX) and fluconazole (FLZ) were used as positive controls.All experiments were conducted in triplicate for each extract.The MIC was the lowest concentration of antimicrobial agent that inhibited observable microbial growth.
Minimum lethal concentration (MLC) was preformed using the standard microdilution method.The MLC was detected as the lowest concentration of S. persica extract or ChNPs that did not allow visible growth (99.9%killing) on the agar plates after the incubation period.All procedures and microbiological manipulations were carried out in a Class II biological safety cabinet.
Based on the findings of antimicrobial tests, the most effective antimicrobial concentration of S. persica (100 mg/ mL = 10%) and the maximum concentration of ChNPs (5 mg/mL = 0.5%) that provide better viscosity have been used to prepare the SChNPs mixture using three formulae and evaluated against all the test strains.Propylene glycol was added as a solvent for the hydroalcoholic extract of S. persica, and l-ascorbic acid was added to acidify the medium to make it suitable for the solubility of ChNPs.In addition, l-ascorbic acid acts as an antioxidant to protect the preparation contents against oxidation (Table 1).

Scanning electron microscope (SEM) evaluation
The most sensitive bacteria (S. pneumonia) was exposed to S. persica, ChNPs, and SChNPs mixture for 24 h.S. pneumonia grown without an extract was taken as a control.After incubation, cells were centrifuged at 5000 r/min for 10 min.The pellets were washed with sterile PBS and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde for 4 h at 4 °C with an intermittent vortex.The cell biomass was fixed to a glass coverslip, and morphological changes in the cell wall were viewed under SEM (JEOL SEM, Filed Emission 7610F).

Fourier-transformed infrared (FTIR)
FTIR spectroscopy (FT-IR, Agilent Technologies, Santa Clara, California, United States) was used to identify the functional groups presented in the S. persica extract, ChNPs, and the SChNPs mixture.FTIR was measured in the range of 4000-500 cm −1 .

Gas chromatography-mass spectrometry (GC-MS)
The phytochemical analysis of S. persica extract and the SChNPs mixture was accomplished on GC-MS equipment (PerkinElmer/Clarus500, United States).

Statistical analysis
Results were documented using Microsoft Excel 365, and computations were run three times.The mean and standard deviation are calculated based on the results.The MICs were computed using the SPSS software program, 2001 (version 15.0, Chigao, IL, USA).The antimicrobial efficacy of the different tested compounds was examined using one-way analysis of variance (ANOVA).Results were considered statistically significant when P < 0.05.

Antimicrobial testing
The findings showed the antimicrobial activity of S. persica extract and ChNPs (Fig. 1), this activity was concentration dependent.S. persica extract at 100 mg/mL was the most effective against all tested strains.The highest inhibition was observed against S. pneumonia and S. mutans.Overall, S. persica demonstrated more pronounced antimicrobial activity (inhibition zones ranged from 12.8 to 2.0 mm) against gram-positive strains (S. pneumonia, S. mutans, and S. aureus) than gram-negative strains (K.pneumonia, P. aeruginosa, and E. coli), where the inhibition zones ranged from 9.6 to 1.1 mm.In addition, S. persica extracts were effective against both tested candida Table 1.Formulae of the S. persica, ChNPs, and the SChNPs mixture.species.The inhibition zones ranged from 9.3 to 1.6 mm in accordance with the S. persica concentration.With the 100 mg/mL concentration, C. albicans was most susceptible, followed by C. krusei (Table 2a).ChNPs at 100 mg/mL were the most effective concentration against all tested microbial strains.The highest inhibition was observed against S. pneumonia, E. coli, K. pneumonia, and both strains of fungi.Overall, ChNPs demonstrated pronounced antimicrobial activity, with inhibition zones ranging from 11.2 to 5.8 mm against gram-positive strains and gram-negative strains.A similar inhibition zone (11.5-6.9 mm) was observed against both tested candida species (Table 2b).
The findings of MIC and MLC showed that S. pneumonia and both fungal strains were the most sensitive to the S. persica extract (6 and 12.5 mg/ml) and ChNPs (25 and 50 mg/ml), respectively (Table 2c).
The antimicrobial efficacy of the mixture (SChNPs) of 10% S. persica and 0.5% ChNPs against tested bacterial and fungal strains is presented in Table (2d).In all tested strains, the prepared mixture showed a significant zone of inhibition with a clear decrease in MIC.The range of the MIC was determined between 4 and 32 mg/ mL.The maximum reduction was observed with S. mutans and S. pneumonia at 4 and 8 mg/mL, respectively.Additionally, it displayed good activity against S. aureus and E. coli, with MIC values of 16 mg/mL.Moreover, it exhibits good efficacy against K. pneumonia and P. aeruginosa, with MIC values of 32 mg/mL.The SChNPs mixture also showed equivalently high activity against both fungal strains tested (C.albicans and C. krusei), with MIC values of 8 mg/mL.

Scanning electron microscope evaluation
Scanning electron microscopy images of S. pneumonia treated with SChNPs showed some morphological changes as compared to the control (untreated S. pneumonia cells).Normal S. pneumonia cells were smooth, intact, and regular cocci (Fig. 2a), while ChNP-treated S. pneumonia showed slight irregularity in shape and aggregation (Fig. 2b).S. persica-treated cells showed minute irregularities in shape and clustering (Fig. 2c).Whereas SChNPstreated cells showed remarkable morphological changes like blebs and destructed cells (Fig. 2d).

Fourier-transform infrared spectroscopy (FT-IR)
The FT-IR spectra peak of S. persica, ChNPs, and the SChNPs mixture are shown in Fig. 3a-c, and their probable functional groups are presented in Table 3.The spectrum shows many biologically active functional groups.FTIR spectrum results show the presence of aldehydes, alkenes, carboxyl nitrogen-containing group, aromatic primary amines, sulfoxides, amines, esters, ethers, and alcohols in both samples.However, some additional bands for ketones, carboxylic acids, and benzenes were observed in the SChNPs mixture.These functional groups mainly belong to many secondary metabolites such as phenols, alkaloids, flavonoids, saponins, terpenoids, and polyphenol.

Gas chromatography-mass spectrometry
GC-MS chromatogram analysis of S. persica, and the SChNPs mixture is shown in Fig. 4 (a and b).Table 4a and b represent the list of major compounds identified by their retention time, molecular formula, and molecular weight.

Discussion
Nowadays, many plant-derived herbal medicines are being used in oral health care, mainly as antimicrobial, anti-inflammatory, and analgesic agents.Mouthwashes derived from medicinal plants have shown promising results in plaque and gum diseases due to the presence of active ingredients with antioxidant properties 22 .In this study, the ethanolic extracts of S. persica exhibited concentration-dependent antimicrobial activities against all tested strains, and the highest inhibition was observed against S. pneumonia and S. mutans.Several reports have also found extracts of S. persica to be effective antimicrobial agent 23 .Overall, the tested ethanolic extract demonstrated more pronounced antimicrobial activity against gram-positive strains than gram-negative strains.This difference is probably caused by the bacterial cell walls having different chemical compositions, especially in the outer membrane of lipopolysaccharides (LPS) structure 24 .LPS are large molecules consisting of a lipid and a polysaccharide that create a barrier to diffusion, making gram-negative bacteria more resistant to antimicrobials.
Nano-based dental materials are increasingly used clinically for oral hygiene due to their antimicrobial activity 19 .Chitosan is used in many commercial and dental products, such as toothpaste, enamel repair materials, dental restorative materials, coatings on implants, and adhesives 25 .
In this study, the antimicrobial activity of the SChNPs mixture, consisting of 10% S. persica ethanolic extract, 0.5% ChNPS, 1% l-Ascorbic acid, and 10% propylene glycol, was evaluated against the previously mentioned standard strains, and a synergistic effect was observed through the widening of the inhibition zone.The presence of different bioactive phytochemical compounds in the SChNPs mixture accounted for the broad-spectrum antimicrobial activities.As a result of the existence of bioactive compounds like alkaloids, tannins, flavonoids, phenols, terpenoids, and anthraquinones in the SChNPs mixture, broad-spectrum antimicrobial activities were detected.There are several possible mechanisms for how some secondary metabolites act against microorganisms: alkaloids may disrupt cellular walls and/or inhibit DNA replication 26 ; tannins may deactivate microbial adhesions, enzymes, and transport proteins in cellular envelopes 27 ; flavonoids may inhibit the synthesis of nucleic acids and energy metabolism of microbial cell membranes 28 ; phenolic compounds and terpenoids may disrupt the microbial cell membranes 29 ; anthraquinones act by increasing the superoxide anion levels and increasing the amount of oxygen molecules 30 .The antimicrobial properties of chitosan are believed to function through binding to negatively charged bacterial cell walls, altering membrane permeability, followed by attachment to DNA, inhibiting DNA replication, and finally causing cell death 31  www.nature.com/scientificreports/ The MLC/MIC ratio for the SChNPs mixture was determined to define whether the pharmaceutical formula was bacteriostatic or lethal in the tested samples.It is generally considered that a MLC/MIC ratio over 4 equates to bacteriostatic effects, while lesser values indicate lethal effects 32 .Consequently, the SChNPs mixture showed lethal efficacy against S. mutans, S. pneumonia, C. albicans, and C. krusei ; but bacteriostatic effects on S. aureus, E. coli, K. pneumonia, and P. aeruginosa.
The effects of S. persica extract and ChNPs on the surface morphology of S. pneumonia were examined by SEM.No morphological changes were observed in untreated S. pneumoniae cells with clear cell wall structure; however, the results showed a special trend of changes in cells treated with S. persica extracts, ChNPs, and the SChNPs mixture.S. pneumonia cells treated with the mixture of 10% S. persica and 0.5% ChNPs exhibited a more irregular cell shape and a high degree of cell lysis as compared to the individual solution.These results explained the synergistic effect through induced morphologic alterations by the damaging bacterial membrane and leaked cytoplasmic material.SEM results are well explained by the occurrence of many active phytocompounds, which were detected by GC-MS analysis.Many studies have reported the antimicrobial activity of these active compounds, such as tetradecanoic acid, pentadecanoic acid, n-Nonadecanol-1 33 , 6-Octadecenoic acid , l-(+)-Ascorbic acid 2,6-dihexadecanoate 34 , Eicosanoic acid ,1-Nonadecene, 9-Octadecenoic acid 35 , which were not detected in S. persica extract alone.These results support the SEM findings that the SChNPs mixture induces maximum damage to the bacterial cells.The biological activity of any compound is influenced by its functional groups, which aid in evaluating the structure-function relationship of the bio-organic compound.FT-IR spectral analysis showed some additional peaks in the SChNPs mixture that were not detected in S. persica extract.FTIR analysis plays a crucial role in identifying and quantifying plant substances such as cell wall components, proteins, and lipids, aiding in understanding biochemical changes in samples.Changes in band height suggest modifications in chemical groups within lipids, carbohydrates, proteins, and cell wall regions 36,37 .

Conclusion
Across all studied strains, the prepared mixture of 10% S. persica and 0.5% ChNPs (SChNPs) demonstrated a large zone of inhibition with a distinct decrease in MIC, indicating its synergistic effect.The use of SChNPs mixture as a mouthwash can act synergistically to control oropharyngeal microbes that are implicated in secondary micorbial infections associated with primary viral infections.

Figure 2 .Figure 3 .
Figure 2. SEM micrograph showing the S. pneumonia cells: without any treatment as regular and smooth cocci (a); with aggregation and various levels of degeneration after 24 h treatment with ChNPs (b); S. persica (c); and the SChNPs mixture (d).

Figure 4 .
Figure 4. GC-MS analysis to detect anti-bacterial compound in S. persica extract (a) and the SChNPs mixture (b).

Table 2 .
Antimicrobial activity profile of ethanolic extract of S. persica (a) and ChNPs (b) at different concentrations against gram-positive, gram-negative bacteria, and fungi (inhibition zone in mm).2a-Strep.Streptomycin, Amph.B Amphotericin B, NI No inhibition, ND not detected.2c-The mean values of MIC and MLC for the ethanol extracts of S. persica and ChNPs.Values are presented as mean ± standard deviation.MIC minimum inhibitory concentration, MLC minimum lethal concentration, ND not detected.2d-Minimal inhibitory concentrations versus cup plate results of S. persica, ChNPs, and the SChNPs mixture against gram-positive, gram-negative bacteria, and fungi.MIA mean of inhibition area, MIC minimal inhibitory concentrations, CHX Chlorohexidine, FLZ Fluconazole, ND not detected.

Table 4 .
Bioactive compounds existing in S. persica extract (a) and the SChNPs mixture (b).